Method and apparatus for analyzing a mixture of sample substances

ABSTRACT

An apparatus for analyzing a mixture of sample substances comprises a fluid-guiding structure wherein the sample substances are moving essentially in one dimension along the structure and are subject to at least two separation mechanisms, such as capillary electrochromatography (CEC) and capillary zone electrophoresis using high voltage. The signals from a detection means are supplied to a signal processing means, which derives a parameter therefrom which in turn is used to derive improved measuring results for each of the separation results associated with the various separation mechanisms, respectively. The apparatus can be used for protein analysis, for example in combination with a mass spectrometer.

[0001] The invention relates to a method and a corresponding apparatusfor analyzing a mixture of sample substances. Such a method can be usedfor identifying chemical or biochemical substances in a mixture and fordetermining the concentration of these substances. The method can beused, for example, for analyzing proteins.

BACKGROUND OF THE INVENTION

[0002] The field of analytical chemistry and biochemistry is concernedwith the measurement of composition, amounts and properties ofsubstances, such as solutions of chemicals, biochemical substances, orother analytes. An important biochemical project that has been completedin the past years is the human genome project. This project has broughtup a new business field, pharmacogenomics, which has the goal tounderstand biological functions and to influence such functions byspecific interventions with pharmaceutical substances. By comparingprotein-expression after drug treatment with protein expression inuntreated condition, it is possible to relate the observed changes tothe effects or function of the drug. Recent works have shown that thereis a lack of correlation between transcriptional profiles and actualprotein levels in cells. Protein analysis has therefore becomeindispensable and complementary to genomic analysis in order to obtainan accurate picture of cellular function and metabolism.

[0003] Methods and apparatus have been developed to enable detection oridentification of analytes in smaller and smaller amounts or under moreuseful conditions. In particular, protein analysis requires speedyexecution and high separation efficiency if applied as a screeningmethod for various cell and tissue types. The established method forprotein expression monitoring is gel electrophoresis on polyacrylamide(PAGE). While this is efficient as a preparative method, it is by fartoo slow for screening applications. It requires a lot of timeinvolvement of an operator, and often the separated spots have to be cutout manually from these preparations so that they can subsequently beanalyzed in a Maldi-TOF-MS system (Maldi: matrix assisted laserdesorption ionization; TOF:time-of-flight; MS: mass spectrometry).

[0004] Often it is difficult to achieve enough separation or peakcapacity in order to detect and identify all individual fragments of asample in just one run. This limitation usually is a reason to usetwo-dimensional separation devices wherein a hybrid of two separationmechanisms is formed. Examples for such two-dimensional separations are:ion exchange/reversed phase liquid chromatography, or isoelectricfocusing (IEF)/SDS-PAGE, which is called two-dimensional gelelectrophoresis (2D-GE). Regarding state of the art, it is referred tothe article, G. L. Corthals et al.: “The dynamic range of proteinexpression: A challenge for proteomic research”, Electrophoresis 2000,21/6, pages 1104-1115; and V. C. Wasinger et al.: “Proteomic tools forbiomedicine”, Journal of Chromatography B, 771 (2002), pages 33-48.

[0005] In the pharmaceutical industry, high throughput screening isoften required. In order to screen for statistically relevantparameters, a large number of analysis steps are performed in parallel.High throughput screening requires automation, speed and reliableoperation. In this approach often all the 2D-GE platforms cannot fulfillthe requirements.

[0006] Several attempts have been made to create an automatedtwo-dimensional separation arrangement. In one approach, liquidchromatography is combined with capillary electrophoresis. In anotherapproach, disclosed in EP-A 977030, a two-dimensional electrophoresisseparation device is implemented on a microchip.

[0007] The mentioned devices and methods have their primary applicationsin the field of proteomics. There is such a broad variety of proteinsthat a simple uni-dimensional separation quickly reaches its limits.Furthermore, the interesting proteins in a sample indicating specificdisorders or diseases often are only present in small quantities,whereas there is a substantially larger portion of proteins in thesample which are not of particular interest. This has the consequencethat small, but interesting peaks in the measuring results are concealedunder tall peaks which are of no particular interest for the actualanalysis, which in turn requires even more peak capacity to get clearer,more distinct readings.

[0008] Reliable results in the field of proteomics are so important thatone even has to take the trouble of several-days measurements in orderto produce a clear distinct two-dimensional pattern. It has been commonto run a two-dimensional gel electrophoresis, cut out the correspondingspots to extract a clean population and then to run it on a Maldi-MS oreven a sequencer, see also Mary F. Lopez: “Better approaches to findingthe needle in a haystack: Optimizing proteome analysis throughautomation”, Electrophoresis, Vol.21, Issue 6 (2000), Pages 1082-1093.

SUMMARY OF THE INVENTION

[0009] It is thus an object of the invention to provide a method and acorresponding apparatus for analyzing a mixture of sample substances,which allows to achieve similar or better separation results as thetwo-dimensional methods of the prior art, and which is easily automatedand which is more suitable for high-throughput operation.

[0010] It is also an object of the invention to provide a method and anapparatus for fast and accurate protein analysis with high resolution.

[0011] According to the invention, these objects are achieved by amethod as defined in claim 1 and by an apparatus as defined in claim 10.The method according to the invention is thus defined by a processwherein the sample substances are moving essentially in one dimensiondefined by a fluid-guiding structure, such as a capillary or a channel,wherein the sample substances are subject to at least a first separationmechanism and a second separation mechanism within said fluid-guidingstructure, wherein the sample substances are detected for providingdetection signals, and wherein at least one parameter is derived fromsaid detection signals, which is used for deriving at least firstseparation results associated with the first separation mechanism andsecond separation results associated with the second separationmechanism.

[0012] It is an underlying idea of the invention to combine severalseparation modes in a way that the final physical separation actuallydoes not have to be perfect, but that with sophisticated detection ofmultiple parameters additional information can be derived allowing thecalculation of clean bands of sample substances. In that way, a reliableidentification of the sample substances is possible, even if the samplesubstances have not been separated completely at the locations wherethey have been detected.

[0013] The invention has the additional advantage that all samplecomponents are moving towards a single physical outlet, which is idealfor interfacing a mass spectrometer or a NMR apparatus. In thetwo-dimensional arrangements of the prior art the sample substances arespread over several axes, which requires a full-area detection, whereasthe invention permits to record the concentration at a specificlocation. In case of a mass spectrometer coupled to an apparatus of theinvention, this location may be the atmospheric pressure ionizationsource (API source).

[0014] In an embodiment of the invention a timing or speed measurementis used to derive additional information. Either the time differencebetween occurrences at different detection locations is derived, or amore complex approach with Fourier detection may be used., Also, aphotodiode array arranged along the fluid-guiding structure may be usedto monitor movement of the bands. A photodiode array as such is known,in a different connection, from EP-A 840113, with the title “MicrochipElectrophoretic Method and Apparatus”. Based on the geometric design ofthe set-up the path of movement of the sample is known. From the actualspeed and total time it is possible to calculate the specific parametersfor each dimension.

[0015] The concept of the invention can also be realized for 3 or moredimensions. Also, the orthogonal measurement parameter does notnecessarily have to be speed or timing. Other parameters can be selectedfrom, but are not limited to, charge state, conductivity, viscosity,temperature, pH, dipole moment, or affinity, if these parameters areindicative of a significant factor in the separation in either of theanticipated dimensions.

[0016] The invention thus provides a mechanically simple apparatus whichoperates in one dimension, but which allows to derive separationinformation, which would otherwise only be accessible by atwo-dimensional separation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In the following embodiments of the invention will be explainedwith reference to the drawings.

[0018]FIG. 1 is a schematic diagram of a first embodiment of theinvention.

[0019]FIG. 2 shows the detector output signals as a function of time inthe embodiment of FIG. 1.

[0020]FIG. 3 is a graphical representation of concentration vs. mobilityand retention factor as derived in the embodiment of FIG. 1.

[0021]FIG. 4 is a schematic diagram of a second embodiment of theinvention.

DETAILED DESCRIPTION

[0022] In the embodiment of the invention shown in FIG. 1, a capillary 1is packed with packing material 2, and the ends of the capillary areconnected to a high voltage power supply 3, respectively. The packingmaterial 2, which fills the left portion of the capillary 1, is of thetype, which is used, in capillary electrochromtography (CEC). Samplesubstances, such as proteins, which have been introduced into thecapillary, are moved through the capillary by means of the electricfield provided by the power supply 3. The movement due to the electricfield is dependent on the mobility of the sample substances,respectively. Since there is an interaction between the samplesubstances and the packing material 2, there is also retention in thecapillary which is dependent on the specific sample substance. Thus,there are combined separation modes in the capillary, i.e., retentionand electromigration.

[0023] A first detector 11 is arranged near the end of the packing 2 anda second detector 12 is arranged near the end of the capillary 1. Thedetectors may be of any type, for example absorbance detectors may beused for protein analysis, fluorescence detectors may be used for DNAanalysis. Other detectors, such as conductivity detectors, may also beused for specific applications. In the embodiment shown, the twodetectors are of the same type, but it is also possible to use detectorsof different types.

[0024] In FIG. 1, the detectors are only shown schematically. The lines11 a and 12 a designate the locations in the capillary where samplesubstances are detected. The output signals of the detectors 11 and 12are supplied to a data processing unit 13, which derives more refinedmeasuring results from the two input signals in a way which will bedescribed below.

[0025]FIG. 1 shows as an example two-separated sample substances 8 and9. These two sample substances pass the first detector 11 at differenttimes t1 and t2, respectively. This is illustrated in FIG. 2, whereinthe detector output signal of detector 11 as a function of time is shownas curve #1, and the detector output signal of detector 12 is shown ascurve #2. The area of the peaks is a measure of the sampleconcentration. After the substances 8 and 9 have been detected by thedetector 11 they are moving to the detector 12 under the influence ofthe electric field in the capillary, without retention taking place. Thesubstances 8 and 9 are then measured by the detector 12 at times t3 andt4, respectively. From the time differences t3−t1 and t4−t2 themobilities of the substances 8 and 9, respectively, are determined bythe data processing unit 13, based on the known electric field strengthin the capillary and on the known distance between the two detectors.

[0026] From the determined mobility values of the sample substances 8,9and from the measurements by detector 11, the retention factors of thesample substances can be determined. The final result is shown in FIG.3, which is a graphical representation of mobilities and retentionfactors for the various sample substances and of the correspondingconcentrations. On the basis of the determined retention factor andmobility of a specific sample substance, a precise identification ofthis sample substance is possible, for example by using data bases ofknown mobilities and retention factors.

[0027] In capillary electrophoresis, there is typically a phenomenoncalled electroosmotic flow (EOF) which is more or less pronounceddepending on factors such as the material of the inner capillary walletc. If EOF occurs in an apparatus of the invention (FIG. 1, referencenumeral 7), it does not contribute to the separation of samplesubstances, but it still has an influence on the times when peaks arriveat the point of detection. The EOF, however, can be measured and takeninto account when determining the mobilities of the sample substances,for example by providing known marker substances in the liquidtransported through the capillary, see, for example U.S. Pat. No.5,316,630, with the title “Methods for Chromatography Analysis”. In anembodiment of the invention, one may also use an off-line approach ofthe type described in U.S. Pat. No. 5,009,760, with the title “MeasuringElectrokinetic Properties”, or an on-line approach of the type describedin U.S. Pat. No. 4,456,513, with the title “Measuring ElectrophoreticMobility” or in U.S. Pat. No. 5,441,613, with the title “RealtimeMonitoring, Measurement and Control of EOF”.

[0028] A second, more complex embodiment of the invention is shown inFIG. 4. This embodiment is implemented in lab-on-a-chip technologywherein small channels through which liquids can be transported arearranged on a microfluidic chip. The chip can be made, for example, ofglass or plastic material. FIG. 4 shows a main channel 20 and two sidechannels 21,22 on a microfluidic chip. The arrangement is designed tohave two portions, namely a portion 23 for performing isoelectricfocusing (IEF), and a gel-filled sizing portion 24.

[0029] With the help of a high voltage supply 25 sample substances areintroduced into the main channel 20 through the side channel 21 and thenfocused. Different sample substances in the channel are illustrated withreference numerals 26 and 27. After sample introduction and focusing ahigh voltage is applied along the channel 20 with a second high voltagesupply 28. This high voltage mobilizes the bands towards the portion 24,where the gel effects additional separation according to the size of thesample substances.

[0030] A detection arrangement 29 is used to monitor the absorbanceinside the gel to derive information about concentration and speed ofmovement of the sample substances. The detection arrangement comprisestwo or more detection spots 29 a, 29 b, 29 c, etc. The detectionarrangement may, for example, be of the type described in U.S. Pat. No.5,699,157, wherein a channel is irradiated through an optical mask whilethe substances are moving through the channel and wherein the lightdetected with a photodetector is analyzed, e.g. by Fourier analysis.Alternatively, several light sources and several photodetectors arrangedopposite to the light sources, respectively, could be used to determineconcentration and velocity of the sample substances. Such an arrangementis disclosed, in a different connection, in U.S. Pat. No. 5,303,021,with the title “Optical Detection for Capillary Chromatography”. Theoutput signals from the detection arrangement 29 in FIG. 1 are suppliedto a signal processing unit 33 wherein they are further processed toderive improved separation information.

[0031] Two bands that have been separated in the IEF portion 23 maystill arrive at the detector at the same time. But with the knowledge ofthe speed at the point of detection it is possible to calculate at whichpositions these bands started, respectively, i.e. the specific positionsin the original pH gradient. Measuring speed generally can provideinformation about the size of sample substances, but measuring speeds atspecific points in time allows to derive information about the pI(isoelectric point). The pH of a solution in which a particular aminoacid does not migrate under the influence of an electric field is calledthe isoelectric point of that particular amino acid. At its pI a proteinloses its net charge due to a pH-induced change in dissociation stage.

[0032] It is understood that other detection arrangements than thosedescribed in connection with FIGS. 1 and 4 can be used. For example, onecould use an arrangement of photodiodes, such as a photodiode array,along the separation capillary to monitor the movement of samplesubstances.

1. A method of analyzing a mixture of sample substances, wherein thesample substances are moving essentially in one dimension defined by afluid-guiding structure, such as a capillary or a channel, wherein thesample substances are subject to at least a first separation mechanismand a second separation mechanism within said fluid-guiding structure,wherein the sample substances are detected for providing detectionsignals, and wherein at least one parameter is derived from saiddetection signals, which is used for deriving at least first separationresults associated with the first separation mechanism and secondseparation results associated with the second separation mechanism. 2.Method as in claim 1, wherein detection signals are provided at least attwo different detection locations along the fluid-guiding structure,wherein timing information about the points in time of the occurrencesof the sample substances at the at least two detection locations isderived, and wherein said timing information is used to derive saidparameter.
 3. Method as in claim 1 wherein the first separationmechanism is capillary electrochromatography, and the second separationmechanism is capillary zone electrophoresis.
 4. Method as in claim 3,wherein the timing information is used to derive the electrophoreticmobility of the sample substances, and wherein the derivedelectrophoretic mobility is used to derive improved retention factorsfor the sample substances subject to the first separation mechanism. 5.Method as in claim 1, wherein the first separation mechanism isisoelectric focusing, and the second separation mechanism is gelelectrophoresis.
 6. Method as in claim 5, wherein the signals detectedat the detection locations are processed using Fourier analysis. 7.Method as in claim 6, wherein the sample substances comprise proteins.8. Method as in claim 7, wherein the sample substances are analyzed bymass spectrometry after having been detected at the second detectionlocation.
 9. Method as in claim 8 used in high throughput screening. 10.An apparatus for analyzing a mixture of sample substances, comprising: afluid-guiding structure, such as a capillary or channel or a network ofchannels, wherein the sample substances are moving essentially in onedimension along the structure, means to perform a first separationmechanism with the sample substances in the fluid-guiding structure,means to perform a second separation mechanism in the fluid-guidingstructure, detection means arranged along the fluid-guiding structurefor providing detection signals, and signal processing means coupled tothe detection means for deriving at least one parameter from thedetection signals, which is used for deriving at least first separationresults associated with the first separation mechanism and secondseparation results associated with the second separation mechanism. 11.Apparatus as in claim 10, wherein the detection means comprises at leasttwo detectors, and wherein the signal processing means and the at leasttwo detectors are operative to derive timing information about thepoints in time of the occurrences of the sample substances at thedetectors, and are operative to derive from said timing information andfrom the output signals of the detectors improved information about theseparation of the sample substances due to the first and secondseparation mechanisms, respectively.
 12. Apparatus as in claim 11wherein the means to perform a first separation mechanism comprise acapillary electrochromatography packing and high voltage means togenerate an electric field in the fluid-guiding structure.
 13. Apparatusas in claim 11 wherein the means to perform a first separation mechanismcomprises means to perform isoelectric focusing.
 14. Apparatus as inclaim 13 wherein the detection means comprises an absorbance detector.15. Apparatus as in any of the claim 14 wherein the detection meansfurther comprises an array of photodetectors.
 16. Apparatus as in claim15 wherein the fluid-guiding structure is arranged on a microfluidicchip.